Although the radioimmunoassay (RIA) method is technically established as a high sensitivity measurement of protein or nucleic acid, under the present circumstances, it is impossible to change the aforementioned method to one having high sensitivity beyond a 10−16 moles degree besides improvement of the sensitivity of a detection equipment. And by the RIA method, the place of measurement is not only limited to an isotope experimental facility, but the expiration date of reagents will become extremely short and the sensitivity of reagents will be decrease rapidly, because of using the nuclide of a short time. It also has the problem of radioactive waste problem to the method of radiation measurement. Especially the problem of abandonment in the case of using a long lasting nuclide is serious. Therefore, the research and development of the high sensitivity measurement of protein or nucleic acid have been developed considering “non-radioactive” as a keyword recently. Thus most of the research and development and the technical improvement about the RIA method is not performed in the actual conditions.
A high sensitivity measurement replaced with the RIA method, which is enzyme immunoassay (ELISA method) in the measurement of protein (FIG. 1), and PCR method in the measurement of nucleic acid. The enzyme immunoassay can be proceed to high sensitivity (10−15 moles) by the fluorescence method and the emitting light method from sensitivity of 10−13 mole by the colorimetric assay in the initial development, and the development and improvement of the exclusive measurement device are also proceeding. However, the sensitivity has been arrived to limit, just the operation of measurement is simple.
Moreover, in the PCR method of the high sensitivity measurement of nucleic acid, it is considered the problem of the detection of specific signal for target molecule, the amplification efficiency and the condition of PCR product arriving at a plateau, the quantification of nucleic acid is difficult strictly.
The enzyme immunoassay using antibody-enzyme complex and the nucleic acid probe assay using a nucleic acid-enzyme complex using thio-NAD cycling assay is already known. (Refer to WO2008/117816 (Patent document 1))